Extremely sensitive optical sensors have been constructed by exploiting an effect known as surface plasmon resonance (SPR). These sensors are capable of detecting the presence of a wide variety of materials in concentrations as low as picomoles per liter. SPR sensors have been constructed to detect many biomolecules including keyhole limpet hemocyanin, .alpha.-fetoprotein, IgE, IgG, bovine and human serum albumin, glucose, urea, avidin, lectin, DNA, RNA, HV antibodies, human transferrin, and chymotrypsinogen. Additionally, SPR sensors have been built which detect chemicals such as polyazulene and nitrobenzenes and various gases such as halothane, trichloroethane and carbon tetrachloride.
An SPR sensor is constructed by sensitizing a surface of a substrate to a specific substance. Typically, the surface of the substrate is coated with a thin film of metal such as silver, gold or aluminum. Next, a monomolecular layer of sensitizing material, such as complementary antigens, is covalently bonded to the surface of the thin film. In this manner, the thin film is capable of interacting with a predetermined chemical, biochemical or biological substance. When an SPR sensor is exposed to a sample that includes a targeted substance, the substance attaches to the sensitizing material and changes the effective index of refraction at the surface of the sensor. Detection of the targeted substance is accomplished by observing the optical properties of the surface of the SPR sensor.
There are two common constructions of an SPR sensor. FIG. 1 illustrates a prism-based SPR sensor 10 that is the most common form of SPR sensors. Sensor 10 includes a disposable slide 20 that is placed on a fixed glass prism 12. Slide 20 is coated with a metal film 16 and sensitizing material 22 is capable of interacting with target substance 18 in sample 21. Before placing slide 20 on prism 12, an operator coats prism 12 with an anti-reflection coating 14, often a fluid, in order to prevent light beam 24 from reflecting before reaching metal-film layer 16.
Light source 28 generates light beam 24 that is incident upon sensor 10. Sensor 10 reflects light beam 24 as light beam 26 received by detector 30. At a specific angle of incidence of light beam 24, known as the resonance angle, a very efficient energy transfer and excitation of the surface plasmon occurs in metal film 16. As a result, reflected light 26 exhibits an anomaly, such as a sharp attenuation, and the resonance angle of sensor 10 can be readily detected. When targeted substance 18 attaches to sensitizing material 22, a shift in the resonance angle occurs due to the change in the refractive index at the surface of sensor 10. A quantitative measure of the concentration of targeted substance 18 can be calculated according to the magnitude of shift in the resonance angle.
A second common form of an SPR sensor, known as grating-based SPR sensor, involves the use of a metal diffraction grating instead of glass prism. FIG. 2 illustrates a grating-based SPR sensor 40 in which substrate 45 is formed to have sinusoidal grooves. In grating-based SPR sensors, the period of the groove profile of substrate 45 typically ranges from 0.4 micrometers to 2.0 micrometers. Thin metal film 42 is formed outwardly from the surface of substrate 45 and comprises any suitable metal such as aluminum, gold or silver. In one embodiment, layer 42 comprises silver having a thickness of approximately 100 nm.
Sensitizing layer 44 is formed outwardly from metal film 42. Sensitizing layer 44 is selected to interact with a predetermined chemical, biochemical or biological substance 18 contained in sample 21. In one embodiment, sensitizing layer 44 comprises a layer of antigens capable of trapping a complementary antibody. Recently, several techniques have been developed for attaching antigens as a receptive material to film 42 such as spin coating with a porous silica sol-gel or a hydrogel matrix. Preferably, sensitizing layer 44 is less than 100 nm thick.
In FIG. 2, light source 28 produces light beam 24 incident upon sensor 40 such that detector 30 receives reflected light beam 26. For grating-based SPR sensors, resonance occurs, and reflected light beam 26 exhibits an anomaly, when a polarization component of light beam 24 is perpendicular to the groove direction of the surface of substrate 45 and the angle of incidence of light beam 24 is appropriate for energy transfer and excitation of the surface plasmon in thin metal film 42.
Grating-based SPR sensors have several distinct advantages over prism-based SPR sensors. For example, the resonance angles of grating-based SPR sensors may be finely tuned by adjusting the groove profile. In addition, grating-based SPR sensors do not require the use of an anti-reflection coating. Grating-based SPR sensors, however, suffer from the fact that the light must propagate through the sample as opposed to prism-based sensors in which the incident light propagates through the prism and strikes the metal film opposite from the sample. Propagation through the sample is disadvantageous because the sample tends to absorb or scatter the incident light. For these reasons, grating-based SPR sensors are ill suited for assaying liquids, such as blood, and are primarily used in gas sensing applications. Furthermore, both of the above-described SPR sensors rely on a highly conducting metallic film to support the surface plasmon resonance. This metal film, however, limits the wavelength of the resonance to the red or infrared region of the light spectrum because at shorter wavelengths the conductivity of even the best metals is not sufficient to generate sharp resonances, thereby resulting in lower sensitivity.
For the reasons stated above, and for other reasons stated below which will become apparent to those skilled in the art upon understanding the present invention, there is a need in the art for an optical sensor having the benefits of grating-based SPR sensor that does not require that the incident light propagate through the sample.